The parathyroid hormone receptor type 1 (PTHR1) is a G protein-coupled receptor (GPCR) that regulates calcium and bone homeostasis. Pathological over activity of this receptor is a frequent complication of multiple diseases (e.g. cancer and chronic renal failure), and may result in bone destruction as well as severe hypercalcemia-induced morbidity. To date, treatment by receptor blockade (i.e. using peptide antagonists which inhibit PTHR1-mediated cAMP production) has proven ineffective. The applicants have observed that the PTHR1 is distinguished from other GPCRs by its substantial level of basal, agonist-independent function. This constitutive activity is difficult to appreciateat the level of second messenger signaling (i.e. cAMP formation) but becomes readily apparent when downstream function is monitored using transcriptional activation as a readout. The applicants therefore postulate that inhibition of both, ligand-induced and constitutive activity is needed for therapeutic efficacy. This cannot be achieved by conventional antagonists but requires a different type of drug, by definition an inverse agonist. The applicants propose to develop a novel HTS assay which is sensitive enough to detect basal PTHR1 activity as well as ligand-induced inverse agonism when screening large libraries of non-peptide compounds. Specific Aim 1 is to develop and optimize a reporter gene assay that monitors inhibition of PTHR1 induced basal transcriptional activity. To achieve this, a stably transfected cell line (co-expressing PTHR1 and a reporter gene) will be generated. Specific Aim 2 is designed to validate this assay in a 384-well format by screening a library of 5,300 compounds from diverse sources; including 1,600 FDA approved drugs (collaboration with the Broad Institute). Once HTS-readiness has been documented, and secondary/tertiary assays for hit validation have been established, the screen will be submitted to the NIH Molecular Libraries Probe Production Centers network for identification of inverse agonists within a 400,000+ small molecule library. The goal of this project is to identify pharmacological probes for further studying the multifacete role of PTHR1 in human physiology, and to define the therapeutic potential of small molecules which block this receptor. PUBLIC HEALTH RELEVANCE: This work will help to identify pharmacological tools for blocking a receptor that is often overactive in cancer and other diseases, and as a result triggers serious complications including bone destruction. No effective blockers have been found in the past. Development of a new method for large-scale compound testing is proposed based on the applicants' discovery that effective blockers should abolish not only hormone-induced receptor activation but also basal receptor-mediated gene transcription when no hormone is present.